Quorum sensing gene cloning - (Mar/02/2007 )

I am new to molecular cloning. I isolated marine bacteria producing quorum sensing signals (acyl-homoserine lactones).I have a skill to screen a colony producing acyl homoserine lactones.I want to clone LuxI and LuxR gene homologues from this bacteria. I have looked at several papers but it was very hard to understand. I have a basic skill and knowledge of molecular cloning through NewEngland Molecular biology summer camp. I don't know what kinds of restriction enzymes I should use etc. Could anyone guide me how to clone LuxI and LuxR homologues? Thanks!

-kawaguchi2001-

-first find LuxI and LuxR homologues. Use your skills with said screen.

-then sequence LuX1 and LuxR homologues. (Since this is a bacterial gene, it should be small and straight forward - probably no introns)

-then cut out your LuxI and LuXR homologues or simply PCR it out, depending which method is more convinient. And move those genes about.

This is the best I can do with the scant bits of infomation provided.

-perneseblue-

QUOTE (perneseblue @ Mar 3 2007, 03:51 AM)

-first find LuxI and LuxR homologues. Use your skills with said screen.

-then sequence LuX1 and LuxR homologues. (Since this is a bacterial gene, it should be small and straight forward - probably no introns)

-then cut out your LuxI and LuXR homologues or simply PCR it out, depending which method is more convinient. And move those genes about.

This is the best I can do with the scant bits of infomation provided.

Thanks! My problem is that I can't find LuxI and LuxR homologues sequence before I make clones because these gene sequence is so divergent among many bacteria species. I couldn't PCR from genomic DNA using PCR primers based on Vibrio Fisherii LuxI and LuxR sequence.

In other words, until I make many E. coli clones to test which colony are producing acyl homoserine lactones I don't know which colony has LuxI and LuxR.

When you don't have not so much knowledge of gene sequence of interest, do you use certain restriction enzymes to cut DNA into pieces, then clone into E.coli? I don't know what restriction enzymes to use.

Once I make many E.coli colonies that has a piece of DNA. I can test which colony is positive.I think that my question is that:When you don't know DNA sequence of gene of interest and only you know how to test positive or negative colonies after making E. coli that has a part of DNA, how do you approach gene cloning of this gene?

Thank you!

-kawaguchi2001-

well here is my go at gene hunting.

Firstly has the genome of your marine bacterium been sequence? y/n

Are there any cDNA libraries of said bacterium? Are there any libraries of any description?

Is anything known about these homologous. Have they been isolated yet? Or partially sequence?

Could you elaborate on what you exactly did to come to conclude that you were not able to find 'LuxI and LuxR homologues'. Did you do a blast search? Did you compare to similar proteins. Did check for conserved seqeuence. Did you do a PCR and try to amplify those conserved sequence. Did you do a southern blot to with nonstringent conditions?

Your assumption is that the marine bacterium LuX1 and LuxR homologous will expressed in a functional form in e coli. This assumption need not be true. Ie even if you have the homologous in e coli, the proteins are not expressed in a functional form. Thus giving a negative signal.

More info would help.

Tentively I say you need to build a library. A genomic DNA library if you are sure the marine bacterium's genes will express in e coli, and your assay sensitive. Or a cDNA library (using RNA obtained when those two quorum sensing genes are active.). Then placing the cDNA library into an expression vector.

-perneseblue-

You are fortunate in that there are already good mechanisms for detecting HSL production. All you need to do is make a library from genomic DNA and select for colonies expressing the LuxI homolog. With luck, the LuxR gene will be nearby. To choose the library construction enzyme, you want to know the GC content of your organism. This will help in selecting a good enzyme. You probably want to cut in 8Kb or so fragments, ideally. You want an enzyme like EcoRI (GAATTC site) which has more A/T than G/C sites if the organism has high GC content. You want an enzyme like BamHI (GGATCC) if the organism has low GC content. You might need to think about methylation issues depending on the organism. I can help with plasmids for HSL detection if you need it. It would help if you have any insight into the size range of the HSL being produced.

-phage434-

Thanks! Perneseblue and phage434,

At this point, I have no knowledge of DNA sequence of this marine bacteria. I guess that I should get 16S rDNA sequenced and the get the identification of this bacteria. How do you find out G+C content?

All I know is that this bacteria produces Acyl-homoserine lactones (AHL-C8 and AHL-C12). I have Agrobacterium NTL4 biosensor for detectiong these AHLs. Since I am analytical chemist, I went to get molecular training to learn molecular cloning. However, it's hard to apply what I learn to our situation. In our lab., we are trying to find new bacteria and even cyanobacteria that produce acyl homoserine lactones and are trying to clone Lux genes.

Other member of our lab. did tried to find conserved region of Lux gene among known bacteria and did PCR on newly found bacteria but it did not work.

You mentioned that LuxR gene may be near the LuxI gene. I just wondered that how do people clone LuxR homologuewithout having assay for LuxR. Maybe, people use antibody to LuxR protein to know that E.coli is expressing LuxR homologue? I am sorry that I am not up to your level of understanding in molecular biology.

Thank you!

-kawaguchi2001-

The genus will often be enough to give a reasonable prediction of GC content. You probably should do a 16S sequence if only to verify that you really have the organism you think you have. You would locate the luxR gene by sequencing the DNA region adjacent to the LuxI gene and hope that it was there. The proteins are likely similar enough that conserved sequence homology will show you have the right protein. Since you already have the Agrobacterium sensor, the next few steps are to grow a culture, isolate genomic DNA (Qiagen Qtip protocol is good, or phenol-chloroform, followed by spooling and resuspension), PCR for 16S and sequencing, trial restriction with different enzymes, and library cloning into a suitable vector (pUC19 is a common choice). I would grow up the raw library overnight and then add a top agar overlay of your biosensor to locate positive colonies. These can be picked and reisolated on fresh plates. It would be good to have a positive control for your sensor -- Sigma makes a collection of AHLs of different lengths, or you can get a LuxI producing strain (I could send you one if you need it).

-phage434-

Phage434,

Thank you for your wonderul advise. When I overlay biosensor on plate with colony and find out positive colony, how do you pick positive colony from there? Or do I need to put each colony in 96 wells microplate to grow in LB broth and then test each supernatant?